Hdac3 deficiency limits periosteal reaction associated with Western diet feeding in female mice

Abstract Diet‐induced obesity is associated with enhanced systemic inflammation that limits bone regeneration. HDAC inhibitors are currently being explored as anti‐inflammatory agents. Prior reports show that myeloid progenitor‐directed Hdac3 ablation enhances intramembranous bone healing in female mice. In this study, we determined if Hdac3 ablation increased intramembranous bone regeneration in mice fed a high‐fat/high‐sugar (HFD) diet. Micro‐CT analyses demonstrated that HFD‐feeding enhanced the formation of periosteal reaction tissue of control littermates, reflective of suboptimal bone healing. We confirmed enhanced bone volume within the defect of Hdac3‐ablated females and showed that Hdac3 ablation reduced the amount of periosteal reaction tissue following HFD feeding. Osteoblasts cultured in a conditioned medium derived from Hdac3‐ablated cells exhibited a four‐fold increase in mineralization and enhanced osteogenic gene expression. We found that Hdac3 ablation elevated the secretion of several chemokines, including CCL2. We then confirmed that Hdac3 deficiency increased the expression of Ccl2. Lastly, we show that the proportion of CCL2‐positve cells within bone defects was significantly higher in Hdac3‐deficient mice and was further enhanced by HFD. Overall, our studies demonstrate that Hdac3 deletion enhances intramembranous bone healing in a setting of diet‐induced obesity, possibly through increased production of CCL2 by macrophages within the defect.

the recruitment of periosteal stem cells to the injury site generates bone-forming osteoblasts within the defect. 4Although periosteal stem cells express chemokine receptors, 5 the study of the mechanisms for recruitment and expansion of periosteal skeletal stem cells to the defect site during intramembranous bone regeneration and how this process is impacted by diet and/or inflammation is less characterized.
1][22] Broad depletion of macrophages, via genetic targeting or clodronate liposomes, attenuates intramembranous bone regeneration. 6Conversely, M-CSF administration enhances the number of macrophages and intramembranous bone formation at the injury site. 6While optimal bone regeneration requires the action of macrophages, the molecular mechanisms by which macrophages promote intramembranous bone healing are less defined.
Osteoclasts likewise play a crucial role during bone healing.Detailed studies of endochondral fracture healing demonstrate that osteoclasts participate in both the early and late phases of fracture healing. 23,24cent evidence also documents that osteoclasts participating in endochondral fracture healing originate from circulating monocytes and/or erythro-myeloid progenitors derived from the spleen. 25,26Osteoclasts are likewise present throughout woven bone during intramembranous bone healing of cortical defects. 6In contrast to endochondral fracture healing, OPG-mediated clearance of osteoclasts during intramembranous bone healing reduced osteoclast number and resorption but did not limit the formation of woven bone within the defect site. 6 the US and other developed countries, consumption of a western diet (e.g.high-fat, high-refined sugar) is linked to adverse health outcomes such as obesity, diabetes and cardiovascular events. 279][30] Moreover, the effects of a western diet on bone mass and fracture healing may be influenced by sex. 31 Our prior work demonstrates that conditional deletion of Hdac3 in myeloid progenitor cells enhances intramembranous bone healing in female mice fed a low-fat/low-refined sugar diet. 21Prior work also well establishes that genetic depletion of Hdac3 in myeloid lineage cells limits inflammatory responses and limits osteoclastogenesis 21,[32][33][34][35][36] ; however, these studies were also performed using mice fed a low-fat/low-sugar diet.In this study, we evaluated the effects of myeloid progenitor-directed ablation of Hdac3 during intramembranous bone regeneration in a model of western diet feeding.

| Generation of Hdac3 conditional knockout mice and diet feeding
Hdac3 fl/fl mice on the C57Bl6/J background 37 were crossed with mice expressing Cre-recombinase under the control of the LysM promoter 38 to delete Hdac3 within Cre-expressing cells.Mice were genotyped for Cre as previously described, 39 or the Hdac3 floxed allele using the following primers: A: 5′-CCACTGGCTTCTCCTAAG TTC-3′, B: 5′-CCCAGGTTAGCTTTGAACTCT-3′ and C: 5′-TTTCCGT ATTT G TG GAAGGA-3′.Hdac3-ablated animals arising from these crossings are referred to as Hdac3 cKO LysM mice.Littermate Cre − animals from crossings were used as controls as appropriate.Animals were housed in an accredited facility under a 12-h light/dark cycle.

| Cortical bone defect generation
Single-cortex, fully stabilized, mid-diaphysis femoral defects were generated in the right femur as previously described. 3,21Mice in all experimental groups were given Buprenorphine SR provided perioperatively at 0.5 mg/kg given 2-4 h before surgery via subcutaneous injection.Mice were then anaesthetised with isoflurane and prepared for aseptic surgery.A small incision was made in the skin overlying the right hamstring and the femoral bone shaft was exposed by blunt dissection of the underlying muscle.A 0.7 mm diameter steel burr drill bit (#19007-07, Fine Science Tools, Foster City, CA) and an electric drill were used to induce a single-cortex defect in the middiaphysis of the femur.Defects were immediately irrigated with 1 mL sterile saline followed by incision closure.Mice were sacrificed by carbon dioxide inhalation on postoperative day 14.

| Micro-computed tomography of the bone defect site
Right femurs were collected 14 days following defect generation and fixed in 10% neutral buffered formalin for 24 h.Femurs were then stored in 70% ethanol prior to scanning using the XT H 225 micro-computed tomography (micro-CT) machine (Nikon Metrology Inc., Brighton, MI).Scans were performed at 120 kV, 61 μA and 720 projections at two frames per projection with an integration time of 708 ms as previously described. 40Scans were performed at an isometric voxel size of 7.11 μm with a 1-mm aluminium filter, 17 minutes per scan.Each scan volume was reconstructed using CT Pro 3D (Nikon Metrology Inc.).Reconstructions were converted to bitmap data sets using VGStudio MAX 3.2 (Volume Graphics GmbH, Heidelberg, Germany).Scans were reoriented via DataViewer (SkyScan, Bruker micro-CT, Kontich, Belgium) to create a new bitmap data set for consistent analysis.Morphometric analysis was performed using SkyScan CT Analyser (CTAn, Bruker micro-CT, Belgium).Bruker's instructions and guidelines for analysis within the field were followed throughout the analysis. 403D analysis of the cortical bone at the defect site was performed in the femoral midshaft approximately 0.7 mm proximal to the growth plate and extending 1.5 mm proximally towards the bone diaphysis.The region of interest (ROI) was defined at the midpoint of the cortical defect, extending approximately 0.35 mm in both directions.The ROI was manually contoured around the cortical bone immediately surrounding the defect site, including the periosteum.Whole bone 3D analysis of this ROI was conducted following subsequent automated contouring.Binary selection of all samples resulted in a global threshold used to separate bone from surrounding tissue within the ROI.3D models of the defect ROI were generated using 3D reconstruction software (CtVox, Bruker micro-CT, Belgium) in a transverse orientation for qualitative analysis.

| Bone histomorphometry
Femurs containing cortical bone defects were decalcified, paraffinembedded and sectioned to a thickness of 7 μm.Sections were TRAP stained using the ELF97 substrate (#387A-1KT, Sigma-Aldrich) and counterstained with DAPI.Serial sections were haematoxylin and eosin stained.The number of TRAP-positive cells within the defect site and the number of osteoclasts per bone perimeter (No. Ocl/B.Pm.) within the defect and within periosteal reaction tissue were quantified as previously described.The percentage periosteal reaction tissue volume at the margins of the cortical defect per total tissue volume was evaluated using ImageJ.

| Isolation and culture of bone marrow macrophages
Bone marrow macrophages (BMMs) were collected from 6-to 8-week-old Hdac3 cKO LysM females or their sex-matched littermates and previously described. 21,39Macrophages were cultured in the presence of 50 ng/mL M-CSF (R and D Systems, 416-ML-050) and treated as per experimental design.

| Osteoblast cell mineralization assays
Calvarial osteoblasts were collected from P7 male or female C57Bl6/J mice as previously described. 39,41Osteoblasts were cultured (0.65 × 10 6 cells/cm 2 ) in a conditioned medium derived from macrophages isolated from Hdac3 cKO LysM or littermate control female mice that was diluted 1:1 in fresh α-MEM and supplemented with 10% FBS, 50 μg/mL of ascorbate, 10 mm β-glycerolphosphate and 1 × 10 7 m dexamethasone.Cultures were fed every 3-4 days with a respective diluted conditioned medium plus osteogenic supplements.Cells were fixed and stained with Alizarin Red on day 10.For blocking experiments, the CCL2 neutralizing antibody (AF-479-NA, R&D Systems) was used at a concentration of 1 μg/mL.

| Wound healing assays
ST2 osteoprogenitor cells were cultured at a density of 5 × 10 5 cells/cm 2 and incubated overnight.A scratch in each cell monolayer (n = 2/group) was made with a 10 μL pipette tip.Cells were then washed twice with PBS and placed in a control or Hdac3 cKO-conditioned medium for 12 h and fixed with neutral buffered formalin.Images were digitally collected using phase contrast microscopy and the area of each wound was assessed using ImageJ software.

| RNA extraction and semi-quantitative PCR
Total RNA was extracted from primary BMMs or calvarial osteoblasts using TRIzol (Invitrogen) and chloroform, and 1 μg was reverse transcribed using the SuperScript III first-strand synthesis system (Invitrogen).The resulting cDNAs were used to assay gene expression via real-time PCR using the gene-specific primers listed in Table 1.Fold changes in gene expression for each sample were calculated using the 2 −ΔΔCt method relative to control after normalization of gene-specific C t values to Ywhaz C t values. 21,39Shown are data from three replicates per group of each genotype with three independent experiments.

| Cytokine arrays
Bone marrow macrophages were collected from 6-to 8-week-old Hdac3 cKO LysM females or their sex-matched littermates.
Macrophages were cultured in the presence of 50 ng/mL M-CSF (R and D Systems, 416-ML-050, Minneapolis, MN) for 3 days and a conditioned medium was collected.Cytokine arrays using the Proteome Profiler Mouse XL Cytokine Array (R and D Systems, ARY028) were then performed in duplicate according to the manufacturer's specifications using the conditioned medium.Spot intensities were determined using ImageJ software and the average intensity was determined.Heatmaps of differentially secreted cytokines were generated using Morpheus analysis software (Broad Institute).

| Immunohistochemistry
For immunohistochemical staining, we utilized the Mouse-and Rabbit-specific HRP/DAB (ABC) IHC Detection (Abcam, #ab236466) according to the manufacturer's specifications.Briefly, sections were deparaffinized in xylenes and a series of graded ethanol and rehydrated in water.Sections were blocked and a primary antibody directed towards CCL2 (ThermoFisher Scientific, #MA5-17040) or an irrelevant control was applied and incubated overnight at 4°C.
Streptavidin polyvalent secondary antibodies and biotin-linked horseradish peroxidase (HRP) were then applied and chromogens were developed with 3,3′-diaminobenzidine.Sections were then briefly counterstained with Fast Green, dehydrated and coverslips were applied with a resinous medium.Digital images of the defect site were collected and the number of positively staining cells per tissue area was determined using ImageJ software.

| Statistics
Statistics were performed in GraphPad Prism (Version 9) using observed between control and Hdac3-ablated female animals fed a high-fat/high-sugar diet (Figure 1C).
Our prior report documents that enhanced bone healing of Hdac3-ablated mice was observed in females, but not males.We confirmed that observation of male mice fed either normal chow or a high-fat/high-sugar diet (Figure 2A,B).We verified that high-fat/ high-sugar diet feeding increased body mass of both control and Hdac3-ablated males (Figure 2C).No differences were observed between control and Hdac3-ablated male animals fed a high-fat/ high-sugar diet.
We noted enhanced bone formation along the periosteal surface of high-fat/high-sugar diet-fed female mice in micro-CT reconstructions.Therefore, we first quantified the amount bone formed along the periosteal surface.Femurs containing cortical bone defects were decalcified, paraffin-embedded and sectioned to a thickness of 7 μm and haematoxylin/eosin stained.We observed enhanced bone formation along the periosteal surface (e.g.periosteal reaction) of control high-fat/high-sugar-fed mice compared to littermates fed a normal chow diet (Figure 3A,B).
Ablation of Hdac3 did not affect the amount of periosteal reaction in normal chow-fed mice but did limit the amount of bone formed along the periosteal surface of high-fat/high sugar-fed animals (Figure 3A,B).We also assessed the amount of periosteal reaction tissue in males and females fed an HFD by removing the amount of bone volume within the defect from the perioseal reaction tissue in our micro-CT analyses (Figure 4).No difference in the percentage of periosteal reaction tissue was noted between control and Hdac3 cKO males (Figure 4).In contrast, control females demonstrated increased periosteal reaction tissue compared to control males (Figure 4).This was mitigated by Hdac3 ablation in females.
These data suggest that myeloid-progenitor-directed deletion of Hdac3 limits unfavourable bone formation in female mice fed a western diet.
We next quantified the number of multinucleated ELF97-positive cells (e.g.osteoclasts) within the defect site and within periosteal reaction tissue.Paraffin sections were TRAP stained using the ELF97 substrate and counterstained with DAPI to visualize multinucleated osteoclasts.We confirmed that Hdac3 ablation limits osteoclast number within the defect site when mice are fed either a normal or a high-fat/high-sugar diet (Figure 5A,B).High-fat/high-sugar diet did not change the number of osteoclasts within the defect of littermate control mice (Figure 5B).Likewise, no change in the number of osteoclasts within periosteal reaction tissue was noted within any group (Figure 5C).

| Macrophages from Hdac3-ablated females produce secreted factors that enhance osteoblast mineralization
Next, we explored mechanisms to explain how myeloid-progenitordirected Hdac3 ablation acts to enhance bone formation.We col-

| Hdac3 deficiency enhances chemokine secretion
To identify potential soluble factors mediating these effects, we performed a cytokine array with a conditioned medium generated from Hdac3 deleted macrophages and controls.As expected from prior reports, we confirmed that Hdac3 enhances the secretion of CHI3L (Chitinase 3 like 1) by macrophages (Figure 7A).In addition, we demonstrate that the chemokines CCL2 (C-C chemokine ligand) and CCL6, as well as GDF-15 (growth and differentiation factor-15) are enriched in the conditioned medium from Hdac3ablated macrophages (Figure 7A).We confirmed by qPCR that Ccl2 expression is elevated by Hdac3-deficient macrophages (Figure 7B,C).

| CCL2 neutralization limits the effects of Hdac3 deletion
We then examined the requirement of secreted CCL2 to mediate enhanced bone formation induced by a conditioned medium derived from Hdac3-ablated macrophages.Although a conditioned medium from Hdac3-ablated macrophages enhanced Alizarin Red staining of calvarial osteoblasts, this was attenuated through cotreatment with a CCL2 neutralizing antibody (Figure 7D).Likewise, increased expression of Alpl, Bgn, Col1a1 and Ibsp was blocked by neutralization of CCL2 within the conditioned medium of Hdac3ablated macrophages (Figure 7E).Lastly, we assayed migration of the osteoprogenitor ST2 cell line and found that a conditioned medium derived from Hdac3-ablated macrophages increased wound healing compared to control a macrophage-conditioned medium (Figure 7F).

| Deletion of Hdac3 elevates Ccl2 expression within the defect
We observed that Hdac3-ablated macrophages produced higher levels of CCL2 and that CCL2 was required for enhanced mineralization of osteoblasts cultured in conditioned medium derived from Hdac3-ablated cells.To determine if this may be true in vivo, we performed immunohistochemistry for CCL2 within the defect

| DISCUSS ION
In the work described here, we evaluated the effects of Hdac3 conditional deletion within myeloid progenitors during intramembranous bone healing in the setting of western diet feeding.We found that high-fat/high-sugar diet feeding enhanced unfavourable bone formation (e.g.periosteal reaction) after injury; thus, Hdac3 ablation limited the amount of periosteal reaction tissue in female mice, but males did not show any enhanced bone regeneration.This was accompanied by decreased numbers of osteoclasts within the defect site of Hdac3-ablated females, but not within the periosteal tissue.
Mechanistically, we show that Hdac3-ablated macrophages produced CCL2 that is required to promote mineralization.
Chemokines are a large family of small, secreted proteins best known to promote chemotaxis and leukocyte infiltration; thus, they play an important role in morphogenesis and wound healing.
CCL2 is a member of the CC-type chemokines.Many cell types, including osteoblasts and various tissue-resident macrophages, express CCL2 to induce monocyte chemotaxis.Genetic depletion of Ccl2 in mice disrupts physiological bone remodelling, resulting in elevated bone mass and diminished osteoclast number. 42,43 contrast, CCL2 facilitates the anabolic actions of intermittent PTH via increasing osteoblast number/activity and the catabolic actions of continuous PTH by enhancing bone resorption. 44CCL2 mediates signalling through the G protein-coupled receptor CCR2.
Prior studies have likewise documented that genetic depletion of Ccr2 (e.g.Ccr2 null mice) reduces the infiltration of macrophages and osteoclasts to fracture callus to limit endochondral fracture healing; furthermore, Ccr2 ablation controls osteoclast activity in an estradiol (E2)-dependent fashion. 45Although prior work describes the actions of Ccl2 in physiological bone remodelling and PTH-induced bone formation, the role of CCL2 during intramembranous bone healing is undefined.Periosteal stem cells express Ccr2, 46 so the enhanced expression of Ccl2 by Hdac3 ablated cells within the defect may promote the recruitment of stem cells to the defect site.
We observed increased bone volume within the defect when control mice were fed HFD chow.9][30] A closer examination of our data revealed that consumption of a high-fat/high-sugar diet increased the percentage of periosteal reaction within control female HFD-fed mice.
These data likewise support that HFD feeding diminishes bone regeneration via suboptimal bone healing.Hdac3 ablation limited this response to high-fat/high-sugar feeding.These effects were limited to female mice.
Oestrogen receptors (ER) α-and β-mediate genomic responses to estrogens.HDAC3 regulates responses to estrogens in a celltype-specific manner.For instance, while one report demonstrated that Hdac3 ablation promoted degradation of Esr1 mRNA (i.e. the gene encoding ERα) in breast cancer cells, 47 others have shown that HDAC3 inhibition promotes Esr1 expression. 48[56] Hdac3 ablation in males does not alter bone healing in our prior published studies, 21 and high-fat/high-sugar feeding did not alter this observation.The effects of Hdac3 ablation could be driven by oestrogen-dependent mechanisms, limiting the effects within males.Alternatively, other isoforms could be functionally compensating for Hdac3 deficiency in males as has been observed in other organ systems. 57,58r study has several important limitations.We chose the LysM-Cre driver to target myeloid progenitor cells due to the broad coverage of cell types involved in bone regeneration, including osteoclasts, macrophages and neutrophils.Prior studies likewise demonstrated that LysM-Cre-mediated deletion of Hdac3 suppressed inflammatory responses and increased regeneration of other tissue types. 32,33,59In contrast, using this approach does not allow us to define the specific cells mediating the enhanced bone healing observed with targeted deletion of Hdac3.We utilized BMMs as our cell source, but isolation of specific macrophage subsets such as osteomacs may refine our understanding of how Hdac3 limits bone healing within females.While mice within our study exhibited significant gains in weight and demonstrated corresponding decreases in weight adjusted cortical bone, we did not observe changes to glucose and insulin responsiveness in tolerance testing.This is in line with prior reports documenting that mice are resistant to the development of diabetes when placed on high-fat/ high-sugar diet.This limitation limits our ability to accurately reflect the full spectrum of changes associated with consumption of a western diet in humans.
In this study, we confirmed that myeloid progenitor-directed ablation of Hdac3 enhances intramembranous bone regeneration in females, but not males.Moreover, we found that Hdac3 deletion protected against unfavourable bone healing associated with consumption of a high-fat/high-sugar diet.We also found that Hdac3-deleted macrophages secreted factors that enhanced mineralization, including CCL2; thus, Hdac3 deficiency may limit periosteal reaction following injury due to increased CCL2-mediated recruitment of periosteal stem cells to the defect site.Periosteal reaction tissue is often disorganized and of lower quality, 30,60 so strategies to limit this type of unfavourable bone formation are animal research was conducted according to guidelines provided by the National Institute of Health and the Institute of Laboratory Animal Resources, National Research Council.The University of Minnesota Institutional Animal Care and Use Committee approved all animal studies.Male and female mice were weaned at 4 weeks old onto either a standard diet or a high-fat/high-glucose diet (Research Diets D12492, 60% fat, 20% protein, 20% carbohydrate.New Brunswick, NJ).Numbers of males and females of each genotype within diet groups are indicated within figure legends.

3 . 1 |
Student's t test or one-way ANOVA as appropriate and post-hoc tests for multiple comparisons when necessary.Specific p values under 0.1 are shown within the figures.Data are shown as box plots from the 25th to 75th percentiles, with whiskers extending to the minimum and maximum value and means shown by horizontal lines or means ± standard deviation.3| RE SULTS Hdac3 ablation limits periosteal reaction in females following western diet feedingHdac3-ablated male and female mice and their control littermates were randomly assigned to either a normal chow diet (NCD) or a high-fat/high-sugar diet (HFD) after weaning.We then generated single cortex, fully stabilized bone defects within the right femoral mid-diaphysis of 12-week-old mice.Micro-CT analyses of the bone defect of female mice 14 days after defect generation revealed that Hdac3 ablation enhanced bone volume at the defect site when comparing cKO (conditional knock out) and littermate control NCD-fed females (Figure1A,B), confirming our prior published observations.21Likewise, HFD-fed controls also demonstrated increased bone volume within the defect compared to NCD-fed control mice (Figure1A,B).Hdac3 ablation enhanced this effect (Figure1A,B).We confirmed that a high-fat/high-sugar diet feeding increased the body mass of both control and Hdac3ablated females (Figure1C).No differences in body weight were F I G U R E 1 Hdac3 ablation enhances intramembranous bone healing of female mice fed a western diet.Hdac3 cKO LysM females and their sex-matched littermate controls were fed a high-fat/high-sugar diet (HFD, Control: n = 10, Hdac3 cKO: n = 9) or normal chow (NCD, Control: n = 10, Hdac3 cKO: n = 9) following weaning.Mice were aged to 12 weeks old and single cortex defects were generated in the femoral midshaft.Two weeks following defect generation, micro-CT analyses were performed.Shown in (A) are reconstructions for femoral cortical defects.(B) Bone volume within the defect, p values as shown.(C) Body weights, p values as shown.

lected
BMMs from 6-to 8-week-old Hdac3-ablated mice and their control littermates.Bone marrow macrophages were cultured in the presence of macrophage colony-stimulating factor (M-CSF) for 3 days to induce macrophage differentiation.Cells were refed with medium containing M-CSF and the conditioned medium was collected after 3 days.Calvarial osteoblasts from C57Bl/6 mice were then collected and cultured in macrophage-conditioned medium for 7 days.Alizarin red staining of calvarial osteoblast cultures demonstrated that soluble factors produced by Hdac3-ablated macrophages enhanced mineralization (+60%, Figure 6A,B).Moreover, a conditioned medium from Hdac3-deficient macrophages enhanced expression of osteoblast phenotypic genes [e.g.alkaline phosphatase (Alpl), biglycan (Bgn), Type I Collagen (Col1a1), Distal-less homeobox 5 (Dlx5), bone sialoprotein 2 (Ibsp) and Osterix (Sp7), Figure 6C].F I G U R E 2 Male mice did not exhibit altered intramembranous bone healing.Hdac3 cKO LysM males and their sex-matched littermate controls were fed a high-fat/high-sugar diet (HFD, Control: n = 9, Hdac3 cKO: n = 10) or normal chow (NCD, Control: n = 9, Hdac3 cKO: n = 10) following weaning.Mice were aged to 12 weeks old and single cortex defects were generated in the femoral mid-shaft.Two weeks following defect generation, micro-CT analyses were performed.Shown in (A) are reconstructions for femoral cortical defects.(B) Bone volume within the defect.(C) Body weights, p values as shown.

F I G U R E 3
Hdac3 ablation limits periosteal reaction in female mice fed a western diet.Hdac3 cKO LysM females and their sex-matched littermate controls were fed a high-fat/high-sugar diet (HFD, Control: n = 5, Hdac3 cKO: n = 5) or normal chow (NCD, Control: n = 5, Hdac3 cKO: n = 5)) following weaning.Mice were aged to 12 weeks old and single cortex defects were generated in the femoral mid-shaft.Two weeks following defect generation, paraffin sections of the femoral defect were collected.(A) Hematoxylin and eosin staining of femoral defects was performed and (B) the percentage of periosteal tissue per defect tissue area was determined.Specific p values are as shown.Scale bar is 100 μm.F I G U R E 4 HFD-fed females form significant periosteal reaction tissues following injury, but males do not.Hdac3 cKO LysM males and females and their sex-matched littermate controls were fed a high-fat/high-sugar diet (HFD, Control: n = 10, Hdac3 cKO: n = 9-10) following weaning.Mice were aged to 12 weeks old and single cortex defects were generated in the femoral mid-shaft.Two weeks following defect generation, micro-CT analyses were performed and the percentage of periosteal bone volume was determined, p values as shown.

F I G U R E 5 F I G U R E 7 F I G U R E 8
site of Hdac3-ablated females and their control littermates.In animals fed a normal chow or high-fat/high-sugar diet, Hdac3 ablation enhanced the number of CCL2-positive cells per tissue area (Figure 8A,B).High-fat/high-sugar diet also increased numbers of CCL2-expressing cells within defect site, which was further enhanced by Hdac3 ablation (Figure 8A,B).These data support that myeloid progenitor directed deletion of Hdac3 enhances CCL2 expression in vivo.Western diet feeding does not alter osteoclast number with intramembranous bone defects.Hdac3 cKO LysM females and their sex-matched littermate controls were fed a high-fat/high-sugar diet (HFD, Control: n = 5, Hdac3 cKO: n = 5) or normal chow (NCD, Control: n = 5, Hdac3 cKO: n = 5)) following weaning.Mice were aged to 12 weeks old and single cortex defects were generated in the femoral mid-shaft.Two weeks following defect generation, paraffin sections of the femoral defect were collected and ELF97/DAPI staining was performed.(A) Representative ELF97 staining within the femoral defect, p values are as shown.(B) Quantification of ELF97-postitive osteoclasts per bone perimeter (No.Ocl/B.Pm.) within the femoral defect.(C) Quantification of ELF97-postitive osteoclasts per bone perimeter (No.Ocl/B.Pm.) within periosteal reaction tissue.F I G U R E 6 Macrophages isolated from Hdac3-ablated females produce soluble factors that enhance osteoblast mineralization.Bone marrow macrophages (BMMs) were collected from 12-week-old female Hdac3 cKO LysM and control littermates.Cells were cultured in the presence of 50 ng/mL of M-CSF for 4 days and conditioned medium (CM) was collected.WT calvarial osteoblasts were cultured in CM derived from female Hdac3 cKO LysM or control littermate BMMs.(A) Alizarin Red staining was performed and (B) Quantified, p value as shown, n = 3. (C) Expression of Alpl, Bgn, Col1a1, Ibsp, and Sp7 were evaluated qPCR, p values as shown, n = 3. Hdac3 deficiency increases the secretion of CCL2 by macrophages to enhance bone formation.Bone marrow macrophages (BMMs) were collected from 12-week-old female Hdac3 cKO LysM and control littermates.Cells were cultured in the presence of 50 ng/mL of M-CSF for 4 days, and a conditioned medium (CM) was collected.(A) Heatmap of secreted cytokines from Hdac3 cKO LysM and control BMMs.Expression of (B) Hdac3 and (C) Ccl2 by BMMs was evaluated by qPCR, p values as shown, n = 3. (D) WT calvarial osteoblasts were cultured in CM derived from female Hdac3 cKO LysM or littermate control BMMs in the presence and absence of a CCL2 neutralizing antibody, and (E) gene expression of osteoblast markers was evaluated by qPCR, p values as shown, n = 3. (F) ST2 osteoprogenitor cells were cultured in CM derived from female Hdac3 cKO LysM or littermate control BMMs and wound healing assays were performed, p values as shown.Hdac3 deficiency increases CCL2 levels within intramembranous bone defects.Hdac3 cKO LysM females and their sex-matched littermate controls were fed a high-fat/high-sugar diet (HFD) or normal chow (NCD) following weaning.Mice were aged to 12-week-old and single cortex defects were generated in the femoral mid-shaft.Two weeks following defect generation, paraffin sections of the femoral defect were collected and (A) immunohistochemistry using and an anti-CCL2 antibody was performed.The scale bar is 174 μm.(B) The number of CCL2-expressing cells per tissue area was determined, p values as shown, n = 3/group.
significant.Future directions of this research will include examining the cellular mechanisms of the female-specific enhancement of bone healing as well as assessment of factors that compensate for the loss of Hdac3 in males.Likewise, use of class I Hdac inhibitors within tissue-engineering strategies aimed at enhancing bone regeneration are also warranted.AUTH O R CO NTR I B UTI O N S Elizabeth K. Vu: Data curation (equal); formal analysis (equal); writing -review and editing (equal).Ismael Y. Karkache: Data curation (equal); formal analysis (equal); writing -review and editing (equal).Anthony Pham: Data curation (supporting); formal analysis